U.S. patent number 4,171,314 [Application Number 05/861,810] was granted by the patent office on 1979-10-16 for 13-halo and 13-deoxy c-076 compounds.
This patent grant is currently assigned to Merck & Co., Inc.. Invention is credited to John C. Chabala, Michael H. Fisher, Helmut H. Mrozik.
United States Patent |
4,171,314 |
Chabala , et al. |
October 16, 1979 |
13-Halo and 13-deoxy C-076 compounds
Abstract
Derivatives of the C-076 compounds are disclosed wherein the
13-position is unsubstituted or substituted by a halogen atom. The
compounds are prepared by removing the glycosyl groups on the
13-position of the C-076 compounds isolated from the fermentation
broth of Streptomyces avermitilis. This is followed by substitution
of the 13-hydroxy group with a halogen, and subsequent removal of
the halogen. The disclosed compounds are antiparasitic,
anthelmintic, insecticidal and acaracidal agents.
Inventors: |
Chabala; John C. (Westfield,
NJ), Fisher; Michael H. (Bridgewater, NJ), Mrozik; Helmut
H. (Matawan, NJ) |
Assignee: |
Merck & Co., Inc. (Rahway,
NJ)
|
Family
ID: |
25336829 |
Appl.
No.: |
05/861,810 |
Filed: |
December 19, 1977 |
Current U.S.
Class: |
549/264;
536/7.1 |
Current CPC
Class: |
C07H
17/08 (20130101); C12N 1/205 (20210501); C07F
7/1804 (20130101); C07D 493/22 (20130101); A01N
43/22 (20130101); C12P 19/623 (20130101); C12R
2001/465 (20210501) |
Current International
Class: |
A01N
43/02 (20060101); A01N 43/22 (20060101); C12P
19/00 (20060101); C07F 7/00 (20060101); C07F
7/18 (20060101); C07H 17/00 (20060101); C07H
17/08 (20060101); C07D 493/00 (20060101); C07D
493/22 (20060101); C12P 19/62 (20060101); C07D
493/22 () |
Field of
Search: |
;260/343.41 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Mishima et al., Tetrahedron Letters 10, pp. 711-714 1975. .
Journal of Antibiotics 29(6), Jun. 1976, pp. 76-35 to 76-42 and pp.
76-14 to 76-16. .
Derwent Abstract 76268w/46 to Sankyo Co..
|
Primary Examiner: Trousof; Natalie
Assistant Examiner: Fan; Jane T.
Attorney, Agent or Firm: Rose; David L. Westlake; Harry
E.
Claims
What is claimed is:
1. A compound having the formula: ##STR3## wherein the broken line
indicates a single or a double bond; R is halogen or hydrogen;
R.sub.1 is hydroxy and is present only when said broken line
indicates a single bond;
R.sub.2 is n-propyl or sec-butyl; and
R.sub.3 is methoxy or hydroxy.
2. The compound of claim 1 wherein R.sub.2 is n-propyl.
3. The compound of claim 1 wherein R.sub.2 is sec-butyl.
4. The compound of claim 3 wherein R is hydrogen.
5. The compound of claim 4 wherein R is hydrogen; R.sub.1 is a
22,23 double bond; R.sub.2 is sec-butyl; and R.sub.3 is methoxy,
which is 13-deoxy C-076 A1a-aglycone.
6. The compound of claim 4 wherein R is hydrogen; R.sub.1 is
hydroxy; R.sub.2 is sec-butyl; and R.sub.3 is methoxy which is
13-deoxy C-076 A2a-aglycone.
7. The compound of claim 4 wherein R is hydrogen; R.sub.1 is a
22,23 double bond; R.sub.2 is sec-butyl; and R.sub.3 is hydroxy,
which is 13-deoxy C-076 B1a-aglycone.
8. The compound of claim 4 wherein R is hydrogen; R.sub.1 is
hydroxy; R.sub.2 is sec-butyl; and R.sub.3 is hydroxy, which is
13-deoxy C-076 B2a-aglycone.
9. The compound of claim 3 wherein R is chlorine.
10. The compound of claim 9 wherein R is chlorine; R.sub.1 is a
22,23-double bond; R.sub.2 is sec-butyl; and R.sub.3 is methoxy,
which is 13-chloro-13-deoxy-C-076 A1a-aglycone.
11. The compound of claim 9 wherein R is chlorine; R.sub.1 is
hydroxy; R.sub.2 is sec-butyl; and R.sub.3 is methoxy, which is
13-chloro-13-deoxy-C-076-A2a-aglycone.
12. The compound of claim 9 wherein R is chlorine; R.sub.1 is a
22,23 double bond; R.sub.2 is sec-butyl; and R.sub.3 is hydroxy,
which is 13-chloro-13-deoxy-C-076-B1a-aglycone.
13. The compound of claim 9 wherein R is chlorine; R.sub.1 is
hydroxy; R.sub.2 is sec-butyl; and R.sub.3 is hydroxy, which is
13-chloro-13-deoxy-C-076-B2a-aglycone.
Description
BACKGROUND OF THE INVENTION
C-076 is a series of macrolides with potent antiparasitic activity.
The compounds are isolated from the fermentation broth of
Streptomyces avermitilis and the morphological characteristics of
the microorganism as well as the methods employed to isolate the
C-076 compounds are fully described in U.S. patent application Ser.
No. 772,601.
Based on taxonomic studies, the microorganisms capable of producing
these C-076 compounds are of a new species of the genus
Streptomyces, which has been named Streptomyces avermitilis. One
such culture, isolated from soil is designated MA-4680 in the
culture collection of Merck & Co., Inc., Rahway, New Jersey. A
C-076 producing sample of this culture has been deposited in the
permanent culture collection of the Fermentation Section of the
Northern Utilization Research Branch, U.S. Department of
Agriculture at Peoria, Illinois, and has been assigned the
accession number NRRL 8165. A sample of NRRL 8165 has also been
deposited, without-restriction as to availability, in the permanent
culture collection of the American Type Culture Collection at 12301
Parklawn Drive, Rockville, Maryland 20852, and has been assigned
the accession number ATCC 31,267.
The above microorganism is illustrative of a strain of Streptomyces
avermitilis which can be employed in the production of the C-076
compounds. However, such description also embraces mutants of the
above described microorganism. For example, those C-076 producing
mutants which are obtained by natural selection or those producted
by mutating agents including X-ray irradiation, ultraviolet
irradiation, nitrogen mustard or like treatments are also included
within the ambit of this invention.
One example of such an organism is a strain of Streptomyces
avermitilis MA 4848 which was isolated after irradiation with
ultraviolet light of Streptomyces avermitilis MA 4680. A
lyophilized tube and a frozen vial of this culture has been
deposited in the permanent culture collection of the American Type
Culture Collection, and they have been assigned the accession
numbers 31272 and 31271 respectively. Slightly higher fermentation
yields of C-076 have been obtained using this frozen stock as
inoculum.
SUMMARY OF THE INVENTION
This invention is concerned with derivatives of the C-076
compounds. Specifically it is concerned with C-076 derivatives
which are unsubstituted at the 13-position or substituted by a
halogen atom. Thus it is an object of this invention to describe
the 13-halo and 13-deoxy compounds of this invention. It is a
further object to describe processes for the preparation of such
compounds. A still further object is to describe methods and
compositions using such compounds as the active ingredient thereof
for the treatment of parasitic infections. Further objects will be
apparent from a reading of the following description.
DESCRIPTION OF THE INVENTION
The C-076 compounds which are the starting materials for the
compounds of this invention are best described in the following
structural formula: ##STR1## wherein R is the
.alpha.-L-oleandrosyl-.alpha.-L-oleandrosyloxy group of the
structure: ##STR2## and wherein the broken line indicates a single
or a double bond;
R.sub.1 is hydroxy and is present only when said broken line
indicates a single bond;
R.sub.2 is n-propyl or sec- butyl; and
R.sub.3 is methoxy or hydroxy.
With reference to the foregoing structural formula, the individual
C-076 compounds are identified as follows:
______________________________________ C-076 R.sub.1 R.sub.2
R.sub.3 ______________________________________ Ala Double bond
sec-butyl --OCH.sub.3 Alb Double bond n-propyl --OCH.sub.3 A2a --OH
sec-butyl --OCH.sub.3 A2b --OH n-propyl --OCH.sub.3 Bla Double bond
sec-butyl --OH Blb Double bond n-propyl --OH B2a --OH sec-butyl
--OH B2b --OH n-propyl --OH
______________________________________
The compounds of the instant invention are derived from the above
C-076 compounds through a series of compounds commencing with the
removal of the .alpha.-L-oleandrosyl-.alpha.-L-oleandrose side
chain at the 13-position. This reaction produces what is identified
as the "C-076 aglycone" compounds characterized by having a hydroxy
group at the 13-position. The C-076 aglycone compounds are then
halogenated with a suitably reactive benzenesulfonyl halide in the
presence of a base to produce the "13-deoxy-13-halo-C-076 aglycone"
compounds. The halogen is then removed in a reaction with a
trialkyltin hydride to produce the "13-deoxy-C-076 aglycone
compounds."
Thus, the compounds of the instant invention are realized in the
foregoing structural formula wherein R is halogen or hydrogen, and
R.sub.1, R.sub.2 and R.sub.3 have the above meanings.
The reaction conditions which are generally applicable to the
preparation of C-076 aglycone involve dissolving the C-076 compound
in an aqueous non-nucleophilic organic solvent, miscible with
water, preferably dioxane, tetrahydrofuran, dimethoxyethane,
dimethyl formamide, bis-2-methoxyethyl ether and the like, in which
the water concentration is from 0.1 to 20% by volume. Acid is added
to the aqueous organic solvent to the extent of 1.0 to 10% by
volume. The reaction mixture is generally stirred at about
20.degree.-40.degree. C., preferably at room temperature, for from
6 to 24 hours. The products are isolated, and mixtures are
separated by techniques such as column, thin layer, preparative
layer and high pressure liquid chromatography, and other known
techniques.
The acids which may be employed in the above process include
mineral acids and organic acids such as sulfuric, hydrohalic,
phosphoric, trifluoroacetic, trifluoromethanesulfonic and the like.
The hydrohalic acids are preferably hydrochloric or hydrobromic.
The preferred acid in the above process is sulfuric acid.
A further procedure for the preparation of the aglycone compounds
is applicable to all of the C-076 compounds, however, it is
preferred for use on the compounds which contain a 23-hydroxy
group, since some degree of addition to the 22,23-double bond is
noticed in those compounds with the 22,23-unsaturation. For the
preparation of the aglycone, 1% sulfuric acid, by volume, in
methanol at from 20.degree.-40.degree. C., preferably room
temperature, for from 6-24 hours has been found to be
appropriate.
The other acids listed above may also be employed for this process,
at approximately the concentration employed for sulfuric acid.
The above described compounds are isolated from the reaction
mixture and mixtures of compounds are separated using techniques
known to those skilled in this art, and in particular the
chromatographic techniques described above.
The "C-076 aglycone" thus produced is then halogenated to produce
the 13-deoxy-13-halo-C-076 aglycone. The halogenation is most
readily carried out in the presence of a sufficiently reactive
benzenesulfonylhalide compound in the presence of a base. The
presence of electron withdrawing substituents on the
benzenesulfonylhalide is advantageous and o-nitro substitution is
preferred. The reaction is carried out in a non-protic inert
solvent such as a halogenated alkyl compound, preferably methylene
chloride or chloroform. The reactants are combined slowly at an
initial temperature of from -25.degree. to +10.degree. C. to
control any initial exothermic reactions, and are maintained at
this temperature for up to 2 hours. The reaction temperature is
then raised to from about room temperature to the reflux
temperature of the reaction mixture for from 10 minutes to 6 hours.
It is necessary to carry out the reaction in the presence of a
base, preferably an organic amine. It has been found to be
preferable to employ the combination of a 4-diloweralkylamino
pyridine and trialkylamine. It is most preferred to employ
4-dimethylaminopyridine and diisopropylethylamine as bases for the
foregoing reactions. The 13-deoxy-13-halo-C-076 aglycone compounds
are isolated by procedures known to those skilled in this art.
In order to avoid unwanted side-reactions, it is important that, in
those C-076 compounds with a hydroxy group at the 5-position (the
B-series of compounds), and to a lesser extent the 23-hydroxy of
the 2-series of compounds, that said hydroxy group be protected.
The protecting group is ideally one which may be readily
synthesized, will not be affected by the reactions to alter the
13-position substituent, and may be readily removed without
affecting any other function of the molecule. One preferred type of
protecting group for the C-076 type of molecule is the
trisubstituted silyl group, preferably a trialkylsilyl group. One
preferred example is the tert-butyldimethylsilyl group. The
reaction is carried out by reacting the hydroxy compound with the
appropriately substituted silyl halide, preferably the silyl
chloride in an aprotic polar solvent such as dimethylformamide.
Imidazole is added as a catalyst. The reaction is complete pg,7 in
from 1/2 to 24 hours at from 0.degree.-25.degree. C. For the
5-position hydroxy group the reaction is complete in about 1/2 to 3
hours at from 0.degree. C. to room temperature. While the
halogenation reaction is much slower at the 23-position hydroxy
group (the 2-series of compounds), if it is desired to protect that
hydroxy group, the reaction will be complete in about 5 to 24 hours
at from about room temperature to 75.degree. C. This reaction is
selective to the 5- and 23-positions under the conditions above
described, and very little silylation is observed at the
13-position.
The silyl group may be removed after the 13-halogenation or the
reaction may be deferred until after the 13-halo group is removed.
The silyl group or groups are removed by stirring the silyl
compound in methanol containing by a catalytic amount of an acid,
preferably a sulfonic acid such as p-toluenesulfonic acid. The
reaction is complete in about 1 to 12 hours at from 0.degree. to
50.degree. C.
The 13-deoxy-13-halo-C-076 aglycone which may or may not have the
silyl groups protecting the 5-and 23-hydroxy groups is then reduced
to form the 13-deoxy-C-076 aglycone. The preferred reducing agent
is one that will selectively remove the 13-halo group but will
leave the remainder of the molecule untouched. One such reducing
agent is a trialkyltinhydride, preferably tributyltinhydride. In
addition it is preferable to include in the reaction mixture a free
radical initiator since it is believed that the reaction proceeds
through a free radical mechanism (not wishing to be bound by theory
however, other possible mechanisms are not excluded). Acceptable
free radical initiators are varied and include peroxides, such as
dibenzoyl peroxides; thiols in the presence of air; azodialkyl
nitriles such as azobisisobutyronitrile; ultraviolet light; heat
and the like. The reaction conditions will vary depending upon the
type of free radical initiator which is employed. For chemical
initiators the reaction is complete in about 1 to 6 hours at from
35.degree.-120.degree. C. The preferred reaction temperature is
about 85.degree. C. If heat is the initiating agent, higher
temperatures are required, about 100.degree.-200.degree. C. for
from 1-6 hours. If ultraviolet light is employed, lower
temperatures are preferred. Generally the reaction will be complete
in from 1-6 hours at -25.degree. to 50.degree. C. in the presence
of ultraviolet light. The trialkyltinhydride reaction is generally
carried out with no solvent under a blanket of nitrogen or other
inert gas. The tin hydride compound is used in excess and becomes
the solvent. If desired, however, an inert solvent such as benzene,
toluene, xylene and the like could be employed. For obvious
reasons, halogenated solvents cannot be employed. The products are
isolated using procedures known to those skilled in this art.
The novel 13-halo-and 13-deoxy-C-076 compounds of this invention
have significant parasiticidal activity as anthelmintics,
ectoparasiticides, insecticides and acaricides, in human and animal
health and in agriculture.
The disease or group of diseases described generally as
helminthiasis is due to infection of an animal host with parasitic
worms known as helminths. Helminthiasis is a prevalent and serious
economic problem in domesticated animals such as swine, sheep,
horses, cattle, goats, dogs, cats and poultry. Among the helminths,
the group of worms described as nematodes causes widespread and
often times serious infection in various species of animals. The
most common genera of nematodes infecting the animals referred to
above are Haemonchus, Trichostrongylus, Ostertagia, Nematodirus,
Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,
Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria,
Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria,
Toxascaris and Parascaris. Certain of these, such as Nematodirus,
Cooperia, and Oesphagostomum attack primarily the intestinal tract
while others, such as Haemonchus and Ostertagia, are more prevalent
in the stomach while still others such as Dictyocaulus are found in
the lungs. Still other parasites may be located in other tissues
and organs of the body such as the heart and blood vessels,
subcutaneous and lymphatic tissue and the like. The parasitic
infections known as helminthiases lead to anemia, malnutrition,
weakness, weight loss, severe damage to the walls of the intestinal
tract and other tissues and organs and, if left untreated, may
result in death of the infected host. The 13-halo and
13-deoxy-C-076 compounds of this invention have unexpectedly high
activity against these parasites, and in addition are also active
against Dirofilaria in dogs, Nematospiroides, Syphacia, Aspiculuris
in rodents, arthropod ectoparasites of animals and birds such as
ticks, mites, lice, fleas, blowfly, in sheep Lucilia sp., biting
insects and such migrating diperous larvae as Hypoderma sp. in
cattle, Gastrophilus in horses, and Cuterebra sp. in rodents.
The instant compounds are also useful against parasites which
infect humans. The most common genera of parasites of the
gastro-intestinal tract of man are Ancylostoma, Necator, Ascaris,
Strongyloides, Trichinella, Capillaria, Trichuris, and Enterobius.
Other medically important genera of parasites which are found in
the blood or other tissues and organs outside the gastrointestinal
tract are the filiarial worms such as Wuchereria, Brugia,
Onchocerca and Loa, Dracunculus and extra intestinal stages of the
intestinal worms Strongyloides and Trichinella. The compounds are
also of value against arthropods parasitizing man, biting insects
and other dipterous pests causing annoyance to man.
The compounds are also active against household pests such as the
cockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle,
Attagenus sp., and the housefly Musca domestica.
The compounds are also useful against insect pests of stored grains
such as Tribolium sp., Tenebrio sp. and of agricultural plants such
as spider mites, (Tetranychus sp.), aphids, (Acyrthiosiphon sp.);
against migratory orthopterans such as locusts and immature stages
of insects living on plant tissue. The compounds are useful as a
nematocide for the control of soil nematodes and plant parasites
such as Meloidogyne spp. which may be of importance in
agriculture.
These compounds may be administered orally in a unit dosage form
such as a capsule, bolus or tablet, or as a liquid drench where
used as an anthelmintic in mammals. The drench is normally a
solution, suspension or dispersion of the active ingredient usually
in water together with a suspending agent such as bentonite and a
wetting agent or like excipient. Generally, the drenches also
contain an antifoaming agent. Drench formulations generally
contains from about 0.001 to 0.5% by weight of the active compound.
Preferred drench formulations may contain from 0.01 to 0.1% by
weight. The capsules and boluses comprise the active ingredient
admixed with a carrier vehicle such as starch, talc, magnesium
stearate, or di-calcium phosphate.
Where it is desired to administer the C-076 derivatives in a dry,
solid unit dosage form, capsules, boluses or tablets containing the
desired amount of active compound usually are employed. These
dosage forms are prepared by intimately and uniformly mixing the
active ingredient with suitable finely divided diluents, fillers,
disintegrating agents and/or binders such as starch, lactose, talc,
magnesium stearate, vegetable gums and the like. Such unit dosage
formulations may be varied widely with respect to their total
weight and content of the antiparasitic agent depending upon
factors such as the type of host animal to be treated, the severity
and type of infection and the weight of the host.
When the active compound is to be administered via an animal
feedstuff, it is intimately dispersed in the feed or used as a top
dressing or in the form of pellets which may then be added to the
finished feed or optionally fed separately. Alternatively, the
antiparasitic compounds of our invention may be administered to
animals parenterally, for example, by intraruminal, intramuscular,
intratracheal, or subcutaneous injection in which event the active
ingredient is dissolved or dispersed in a liquid carrier vehicle.
For parenteral administration, the active material is suitably
admixed with an acceptable vehicle, preferably of the vegetable oil
variety such as peanut oil, cotton seed oil and the like. Other
parenteral vehicles such as organic preparation using solketal,
glycerol, formal and aqueous parenteral formulations are also used.
The active 13-halo- or 13-deoxy-C-076 compound or compounds are
dissolved or suspended in the parenteral formulation for
administration; such formulations generally contain from 0.005 to
5% by weight of the active compound.
Although the antiparasitic agents of this invention find their
primary use in the treatment and/or prevention of helminthiasis,
they are also useful in the prevention and treatment of diseases
caused by other parasites, for example, arthropod parasites such as
ticks, lice, fleas, mites and other biting insects in domesticated
animals and poultry. They are also effective in treatment of
parasitic diseases that occur in other animals including humans.
The optimum amount to be employed for best results will, of course,
depend upon the particular compound employed, the species of animal
to be treated and the type and severity of parasitic infection of
infestation. Generally good results are obtained with our novel
compounds by the oral administration of from about 0.001 to 10 mg.
per kg. of animal body weight, such total dose being given at one
time or in divided doses over a relatively short period of time
such as 1-5 days. With the preferred compounds of the invention,
excellent control of such parasites is obtained in animals by
administering from about 0.025 to 0.5 mg. per kg. of body weight in
a single dose. Repeat treatments are given as required to combat
re-infections and are dependent upon the species of parasite and
the husbandry techniques being employed. The techniques for
administering these materials to animals are known to those skilled
in the veterinary field.
When the compounds described herein are administered as a component
of the feed of the animals, or dissolved or suspended in the
drinking water, compositions are provided in which the active
compound or compounds are intimately dispersed in an inert carrier
or diluent. By inert carrier is meant one that will not react with
the antiparasitic agent and one that may be administered safely to
animals. Preferably, a carrier for feed administration is one that
is, or may be, an ingredient of the animal ration.
Suitable compositions include feed premixes or supplements in which
the active ingredient is present in relatively large amounts and
which are suitable for direct feeding to the animal or for addition
to the feed either directly or after an intermediate dilution or
blending step. Typical carriers or diluents suitable for such
compositions include, for example, distillers' dried grains, corn
meal, citrus meal, fermentation residues, ground oyster shells,
wheat shorts, molasses solubles, corn cob meal, edible bean mill
feed, soya grits, crushed limestone and the like. The active
13-halo- and 13-deoxy-C-076 compounds are intimately dispersed
throughout the carrier by methods such as grinding, stirring,
milling or tumbling. Compositions containing from about 0.005 to
2.0% by weight of the active compound are particularly suitable as
feed premixes. Feed supplements, which are fed directly to the
animal, contain from about 0.0002 to 0.3% by weight of the active
compounds.
Such supplements are added to the animal feed in an amount to give
the finished feed the concentration of active compound desired for
the treatment and control of parasitic diseases. Although the
desired concentration of active compound will vary depending upon
the factors previously mentioned as well as upon the particular
C-076 derivative employed, the compounds of this invention are
usually fed at concentrations of between 0.00001 to 0.002% in the
feed in order to achieve the desired antiparasitic result.
In using the compounds of this invention, the individual 13-halo-
and 13-deoxy-C-076 components may be prepared and used in that
form. Alternatively, mixtures of two or more of the individual
monosaccharide and aglycone C-076 components may be used, as well
as mixtures of the parent C-076 compounds and the compounds of this
invention.
In the isolation of the C-076 compounds, which serve as starting
materials for the instant processes, from the fermentation broth,
the various C-076 compounds will be found to have been prepared in
unequal amounts. In particular an "a" series compound will be
prepared in a higher proportion than the corresponding "b" series
compound. The weight ratio of "a" series to the corresponding "b"
series is about 85:15 to 99:1. The differences between the "a"
series and "b" series is constant throughout the C-076 compounds
and consists of a sec-butyl group and an n-propyl group
respectively at the 25-position. This difference, of course, does
not interfere with any of the instant reactions. In particular, it
may not be necessary to separate the "b" components from the
related "a" component. Separation of these closely related
compounds is generally not practiced since the "b" compound is
present only in a very small percent by weight, and the structural
difference has negligible effect on the reaction processes and
biological activities.
The C-076 derivatives of this invention are also useful in
combatting agricultural pests that inflict damage upon crops while
they are growing or while in storage. The compounds are applied
using known techniques as sprays, dusts, emulsions and the like, to
the growing or stored crops to effect protection from such
agricultural pests.
The following examples are provided in order that this invention
might be more fully understood; they are not to be construed as
limitative of the invention.
The 13-halo- and 13-deoxy-C-076 derivatives prepared in the
following examples are generally isolated as amorphous solids and
not as crystalline solids. They are thus characterized analytically
using techniques such as mass spectrometry, nuclear magnetic
resonance, and the like. Being amorphous, the compounds are not
characterized by sharp melting points, however, the chromatographic
and analytical methods employed indicate that the compounds are
pure.
EXAMPLE 1
23-O-t-Butyldimethylsilyl-C-076-A2a-Aglycone
200 Mg. of C-076-A2a-aglycone in 2.4 ml. of dry dimethylformamide
is combined with 133 mg. of imidazole and stirred until all the
components are dissolved. 146 Mg. of t-butyldimethylsilylchloride
is added and the reaction mixture stirred at room temperature for
24 hours. The reaction mixture is diluted with ether and washed
five times with water. The combined water washes are extracted with
ether and the combined organic layers washed again with water
followed by a single wash with saturated sodium chloride solution.
The ether layer is concentrated to dryness in vacuo affording 340
mg. of a gold colored oil. Preparative layer chromatography of the
oil on two plates of silica gel eluting with a mixture of 5%
tetrahydrofuran and 5% ethanol in methylene chloride affords 113.2
mg. of 23-O-t-butyldimethylsilyl-C-076-A2a-aglycone, the structure
of which is confirmed by mass spectrometry, and nuclear magnetic
resonance.
EXAMPLE 2
23-O-t-Butyldimethylsilyl-13-Chloro-13-deoxy-C-076-A2a-Aglycone
20 Mg. of 23-O-t-butyldimethylsilyl-C-076-A2a-aglycone is combined
with 0.7 ml. of a methylene chloride solution containing 15 mg. of
4-dimethylaminopyridine and 0.021 ml. (15.5 mg.) of
diisopropylethylamine. The mixture is cooled in an ice bath and a
solution of 0.1 ml. of methylene chloride containing 20 mg. of
o-nitrobenzenesulfonylchloride is added dropwise. The reaction
mixture is stirred for 45 minutes in an ice bath and then for 3
hours at room temperature. Ice chips are added to the reaction
mixture and stirred. When the ice is melted, ether is added to the
mixture and the layers separated. The aqueous layer is again
extracted with ether and the combined organic layers washed twice
with water, dried over magnesium sulfate and evaporated to dryness
under a stream of nitrogen affording 35 mg. of a gold film.
Preparative layer chromatography of the material on a single silica
gel plate eluting with 5% tetrahydrofuran and 5% ethanol in
methylene chloride affords 10.1 mg. of
23-O-t-butyldimethylsilyl-13-chloro-13 -deoxy-C-076-A2a-aglycone,
the structure of which is confirmed by mass spectrometry and 300
MHz nuclear magnetic resonance.
EXAMPLE 3
13-Chloro-13-Deoxy-C-076-A2a-Aglycone
A solution of 10 mg. of
23-O-t-butyldimethylsilyl-13-deoxy-C-076-A2a-aglycone in 1.0 ml. of
methanol containing 1% p-toluenesulfonic acid dihydrate is stirred
at room temperature for 5 hours. The reaction mixture is diluted
with 25 ml. of ethyl acetate, and washed with aqueous sodium
bicarbonate and water. The organic layer is dried and evaporated to
dryness in vacuo affording
13-chloro-13-deoxy-C-076-A2a-aglycone.
EXAMPLE 4
13-Chloro-13-Deoxy-C-076-A2a-Aglycone
20 Mg. of C-076-A2a-aglycone is dissolved in 0.7 ml. of methylene
chloride containing 16 mg. of dimethylaminopyridine and 16.8 mg.
(0.023 ml.) of 4-diisopropylethylamine. The reaction mixture is
cooled in an ice bath and 0.1 ml. of methylene chloride containing
21.5 mg. of o-nitrobenzenesulfonylchloride is added dropwise. The
reaction mixture is stirred in an ice bath for 1 hour and allowed
to warm to room temperature and stirred for 4 hours. Ice is added
and stirred until melted. Ether is added and the layers shaken and
separated. The aqueous layer is extracted with ether and the
organic layers combined, washed three times with water, dried over
magnesium sulfate and evaporated to dryness under a stream of
nitrogen affording 40 mg. of a brown film. Preparative layer
chromatography on silica gel plates eluting with 3% tetrahydrofuran
and 1% ethanol in methylene chloride affords 4.7 mg. of
13-chloro-13-deoxy-C-076-A2a-aglycone which is identified by a
nuclear magnetic resonance and mass spectrometry.
EXAMPLE 5
13-Deoxy-C-076-A2a-Aglycone
80 Mg. of 13-chloro-13-deoxy-C-076-A2a-aglycone is dissolved in 1.5
ml. of tributyltinhydride and 20 mg. of azobisisobutylronitrile is
added. The reaction mixture is heated under nitrogen at 85.degree.
C. for 31/2 hours. The reaction mixture is cooled and placed on a
silica gel preparative layer chromatography plate and eluted with
chloroform affording 110 mg. of a glass. Repeated preparative layer
chromatography on silica gel using methylene chloride with 2%
tetrahydrofuran and 0.07% ethanol as eluent affords 10 mg. of a
white glass which is identified by mass spectrometry and 300 MHz
nuclear magnetic resonance as 13-deoxy-C-076-A2a-aglycone.
EXAMPLE 6
13-Deoxy-23-O-t-Butyldimethylsilyl-C-076-A2a-Aglycone
1 Mg. of
13-chloro-13-deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglycone is
dissolved in 50 microliters of toluene and 100 microliters of
tributyltinhydride and 200 micrograms of azobisisobutyronitrile and
heated at 60.degree. C. for 4 hours. The product is isolated by
direct chromatography on a preparative layer silica gel
chromatography plate eluting with 1.5% tetrahydrofuran in
chloroform affording 13-deoxy
23-O-t-butyldimethylsilyl-C-076-A2a-aglycone which is identified by
mass spectrometry.
EXAMPLE 7
13-Deoxy-C-076-A2a-Aglycone
Following the procedure of Example 3 using
13-deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglycone in place of
23-O-t-butyldimethylsilyl-13-chloro-13-deoxy-C-076-A2a-aglycone,
there is obtained 13-deoxy-C-076-A2a-aglycone.
EXAMPLE 8
5-O-t-Butyldimethylsilyl-C-076-B1a-Aglycone
100 Mg. of C-076-B1a-aglycone is dissolved in 1.2 ml. of anhydrous
dimethylformamide and 46 mg. of imidazole is added followed by 50
mg of t-butyldimethylsilylchloride. The reaction is maintained at
20.degree. C. for 30 minutes and diluted with ether. The mixture is
washed with water, dried and concentrated in vacuo to a colorless
glass. Further purification on a preparative layer chromatography
plate eluting with a methylene chloride, tetrahydrofuran mixture
affords purified 5-O-t-butyldimethylsilyl-C-076-B1a-aglycone.
Following the above procedure, utilizing C-076 B2a aglycone in
place of C-076 B1a aglycone, affords
5-O-t-butyldimethylsilyl-C-076-B2a-aglycone.
EXAMPLE 9
5-O-t-Butyldimethylsilyl-13-Deoxy-13-chloro-C-076-B1a-Aglycone
Following the procedure of Example 2 utilizing
5-O-t-butyldimethylsilyl-C-076-B1a-aglycone in place of
23-O-t-butyldimethylsilyl-C-076-A2a-aglycone, there is produced
5O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076 B1a-aglycone.
Following the above referenced procedure using
5-O-t-butyldimethylsilyl-C-076-B2a-aglycone in place of
5-O-t-butyldimethylsilyl-C-076-B1a-aglycone, there is obtained
5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B2a-aglycone.
EXAMPLE 10
5-O-t-Butyldimethylsilyl-13-Deoxy-C-076-B1a-Aglycone
Following the procedure of Example 5 utilizing
5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone in
place of 13-chloro-13-deoxy-C-076-A2a-aglycone, there is produced,
5-O-t-butyldimethylsilyl-13-deoxy-C-076-B1a-aglycone.
Following the above referenced procedure using
5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B2a in place of
5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone,
there is produced
5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone.
EXAMPLE 11
13-Deoxy-C-076-B1a-Aglycone
A solution of 13 mg. of
5-O-t-butyldimethylsilyl-13-deoxy-C-076-B1a-aglycone in 1.0 ml. of
methanol containing 1% p-toluenesulfonic acid dihydrate is stirred
at 20.degree. C. for 3 hours. The reaction is diluted with 30 ml.
of ethyl acetate, washed with aqueous sodium bicarbonate solution
and then with water. The organic layer is dried and evaporated to
dryness in vacuo to afford 13-deoxy-C-076-B1a-aglycone as a clear
glass.
Following the above procedure, utilizing
5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone in place of
5-O-t-butyldimethylsilyl-13-deoxy-C-076-aglycone, there is obtained
13-deoxy-C-076-B1a-aglycone.
If the products of Example 9 are hydrolyzed according to the
foregoing procedure, there will be obtained
13-chloro-13-deoxy-C-076-B1a-aglycone and
13-chloro-13-deoxy-C-076-B2a-aglycone.
EXAMPLE 12
13-Chloro-13-Deoxy-C-076-A1a-Aglycone
Following the procedure of Example 4, employing C-076 A1a-aglycone
in place of C-076 A2a-aglycone, there is produced
13-chloro-13-deoxy-C-076-A1a-aglycone.
EXAMPLE 13
13-Deoxy-C-076-Ala-Aglycone
Following the procedure of Example 5, employing
13-chloro-13-deoxy-C-076-A1a-aglycone in place of
13-chloro-13-deoxy-C-076-A2a-aglycone, there is produced
13-deoxy-C-076-A1a-aglycone.
PREPARATION 1
A 250 ml. baffled Erlenmeyer flask containing 50 ml. of the
following medium:
______________________________________ Lactose 2.0% Distiller's
solubles 1.5% Autolyzed yeast, Ardamine pH 0.5% pH - before
sterilization 7.0 ______________________________________
is inoculated with the contents of one frozen vial of Streptomyces
avermitilis MA 4848 and incubated on a rotary shaker at 28.degree.
C. for 24 hours at 150 RPM.
10 Ml. of the above fermentation media is employed to inoculate 500
ml. of the same medium as above in a 2 liter baffled Erlenmeyer
flask. The fermentation media is incubated at 150 RPM on a rotary
shaker at 28.degree. C. for 24 hours.
All of the foregoing media is employed to inoculate 467 liters of
the following media in a 756 liter stainless steel fermentor:
______________________________________ Lactose 2.0% Distiller's
solubles 1.5% Autolyzed yeast, Ardamine pH 0.5% Polyglycol 2000
0.32 ml./liter pH - before sterilization 7.0
______________________________________
The fermentation media is incubated at 28.degree. C. for 40 hours
with an air flow 10 cubic feet per minute and an agitation rate 130
RPM.
230 Liters of the above media is employed to inoculate 4,310 liters
of the following medium in a 5,670 liter stainless steel
fermentor:
______________________________________ Dextrose 4.5% Peptonized
milk 2.4% Autolyzed yeast, Ardamine pH 0.25% Polyglycol 2000 2.5
ml./liter pH - before sterilization 7.0
______________________________________
The fermentation continues for 144 hours at 26.degree. C. with an
air flow rate of 54.3 cubic feet per minute and agitation of 120
RPM.
The fermentation media are filtered and the mycelial filter cake
washed with about 550 liters of water, the filtrate and washings
are discarded. The filter cake is agitated with about 1500 liters
of acetone for about one hour and filtered. The filter cake is
washed with a mixture of about 150 liters of acetone and 40 liters
of deionized water affording about 2000 liters of extract.
The foregoing fermentation and extraction is repeated on the same
scale affording a further 2000 liters of acetone extract which is
combined with the first extract and evaporated to a volume of about
800 liters. The pH of the concentrate is adjusted to about 4.7 with
concentrated hydrochloric acid and combined with about 800 liters
of methylene chloride. The combined solvents are agitated for about
4 hours and separated. The aqueous layer is combined with an
additional 800 liters of methylene chloride and agitated for about
4 hours. The layers are separated and each methylene chloride
extract separately treated with about 10 kilograms of Super-Cel and
filtered. Both extracts are evaporated to a combined volume of
about 60 liters.
PREPARATION 2
The 60 liter solution of C-076 in methylene chloride of the
previous example is concentrated to dryness in vacuo and the
residue is combined 3 times with 60 liter portions of methanol and
evaporated to dryness to remove any residual methylene chloride.
The final methanol concentrate volume is approximately 36 liters.
The methanol solution is stored overnight and filtered. The filter
cake is washed with 40 liters of fresh methanol and the methanol
filtrates and washings are combined. The methanol solution is
combined with 95 liters of ethylene glycol and 130 liters and
heptane. The 2 layer solution is agitated for 5 minutes and the
lower layer (ethylene glycol and methanol) is separated. The
heptane solution is washed with a mixture of 20 liters of ethylene
glycol and 6.3 liters methanol. After five minutes of agitation,
the lower layer is separated and combined with the first ethylene
glycol/methanol extract. An equal volume of water (approximately
150 liters) containing 79 g. of salt per liter is added to the
ethylene glycol/methanol extracts. This solution is extracted with
150 liters of ethyl ether with agitation for 5 minutes. The ether
layer is washed with 75 liters of water (1/2 volume) and agitated
for 5 minutes and the layers separated. This procedure is repeated
an additional 2 times (the final water wash contains 20 g. of salt
per liter) affording a final ether layer volume of 110 liters. The
ether layer is concentrated in vacuo, to a minimum volume, keeping
the temperature less than 25.degree. C. 40 liters of methylene
chloride is added to the residue and the solution is evaporated to
dryness. This procedure is repeated and the final residue
concentrated in vacuo at 50.degree. C. to dryness.
PREPARATION 3
A 30 centimeter diameter column is prepared with a layer of 34
kilograms of activated alumina followed by a layer of 34 kilograms
of activated carbon in a solution of methylene chloride. The
residue from the previous example is dissolved in methylene
chloride to a volume of 34 liters and applied to the column and
eluted with 34 liters of methylene chloride. these fractions are
discarded. A 3% solution of isopropanol and methylene chloride
(20.8 liters of isopropanol and 660 liters of methylene chloride)
is applied to the column and eluted in approximately 200 liter
fractions. The combined isopropanol and methylene chloride
fractions are evaporated in vacuo at a bath temperature of about
60.degree. C. to a volume of about 20 liters. The bath temperature
is reduced to about 45.degree. C. and the extract is evaporated to
dryness in vacuo. The residue is dissolved in 10 parts methylene
chloride, 10 parts hexane and one part methanol to a final volume
of 15 liters. This solution is applied directly to the Sephadex
LH-20 column of the next example.
PREPARATION 4
A 30 centimeter diameter column is prepared in methanol with 36
kilograms of Sephadex LH-20 (available from Pharmacia Fine
Chemicals, 800 Centennial Avenue, Piscataway, New Jersey 08854) and
washed with a solvent consisting of 10 parts methylene chloride, 10
parts hexane and one part methanol. One-fourth of the C-076
solution of Example 3 is applied to the column and the column
eluted at a rate of 250 ml. per minute. Two 20 liter forecuts are
collected and discarded followed by 20 two liter rich cuts
(identified as fractions 1-20), followed by a single 20 liter tail
cut, which is discarded. Fractions 1-8 are found to contain the
C-076 A compounds and fractions 9-20 are found to contain the C-076
B compounds.
PREPARATION 5
The process of Preparation 4 is repeated on the same scale three
more times and all of the fractions containing the C-076 B
components (fractions 9-20) are combined and evaporated to dryness,
affording 818 g. of crude mixed C-076 B components. The sample is
found to contain 55% C-076 B1 and 39% of C-076 B2. 680.5 G. of this
sample is dissolved in 2 liters of methylene chloride and placed in
a 22 liter three neck round bottom flask followed by the addition
of 13.6 liters of methanol. The methylene chloride is removed by
distillation. 13.6 Liters of ethylene glycol is added as the
methanol is being distilled under reduced pressure. The rate of
distillation is maintained such that the temperature of the
solution did not go below 65.degree. C. When the addition of the
ethylene glycol is complete, the solution is allowed to cool at
5.degree. C. for sixteen hours. The crystals are filtered and
washed with 1 liter of cold ethylene glycol. The crystals are then
redissolved in 2 liters of methylene chloride the solution placed
in a 22 liter three necked round bottom flask. The procedure
described above is repeated twice. The first time 12.5 liters each
of methanol and ethylene glycol is employed and the second time
13.6 liters each of methanol and ethylene glycol is employed. The
final crystals are washed with 1 liter of cold ethylene glycol and
1 liter of water. The crystals are dissolved in 4 liters of water
and dried by filtering through sodium sulfate. The benzene solution
is concentrated to a volume of 2 liters and lyophilized affording
241.2 gm. of a white powder consisting of 98% C-076 B.sub.1 and 1%
of C-076 B.sub.2.
The mother liquors (22 liters) from the first two crystallizations
above are combined and diluted with 22 liters of water. The aqueous
solution is extracted with 60 liters of toluene and again with 15
liters of toluene. The toluene extract is then washed with 48
liters of water. The organic phase is filtered through Super-Cel to
remove any residual water and evaporated affording 336 gm. of solid
material consisting of 79% C-076 B.sub.2 and 16% C-076 B.sub.1
compounds.
PREPARATION 6
In the four Sephadex LH-20 columns of the procedure of Preparation
4, fractions 1-8 contain the C-076 A compounds and are combined. By
HPLC analysis the mixture is found to contain 252 g. of C-076 A2a,
16 g. of A2b, 94 g. of A1a and 24 g. of A1b. The material is
dissolved in a solvent system consisting of hexane:toluene:methanol
in the proportion of 6:1:1 and applied to the Sephadex LH-20 column
of the same dimensions as the one used in Preparation 4 in the
above solvent. Fractions are collected at the rate of 250 ml. per
minute and a 20 liter forecut is collected and discarded. Further
elution affords 2 additional 20 liter forecuts which are also
discarded and 50 four liter rich cuts which contain C-076 A
compounds. Fractions 3-8 are found to contain predominately C-076
A1 components (40.2 g. A1a and 6.7 g. A1b), and fractions 29-36 are
found to contain C-076 A2 compounds (117.2 g. A2a and 7.35 g. of
A2b). Fractions 9-28 contain a mixture of C-076 A1 and A2
compounds.
PREPARATION 7
A sample of 150 g. of C-076 B1 from Preparation 5 is dissolved in 3
liters of a solvent mixture of hexane:toluene:methanol in the ratio
of 3:1:1. The solution is passed through a column of Sephadex LH-20
(of the same dimensions as the one used in Preparation 4) in the
above solvent taking fractions at the rate of 250 ml. per minutes.
After two 20 liter portions of the solvent mixture are collected
and discarded, forecut of 10 liters is taken and discarded. Then 30
richcuts of 2 liters each are taken. Fractions 1-13 and 25-30 are
discarded. Fractions 14-16 are combined and contain 80 g. of
predominately C-076 B1a. Fractions 22-24 are combined and contain
6.7 g. of predominately C-076 B1b. Fractions 17-21 contain a
mixture of C-076 B1a and B1b.
Fractions 17-21 above are combined and concentrated and passed
through a Sephadex LH-20 column with the same solvent system as
above. Three 20 liter forecuts are taken and discarded. Richcuts
are then taken as follows: 5 cuts of 2 liters each (fractions 1-5);
20 cuts of 1 liter each (fractions 6-25); and 10 cuts of 2 liters
each (fractions 26-35). Fractions 1-15 are discarded; fractions
16-21 contain 13.5 g. of C-076 B1a and 0.4 g. of C-076 B1b;
fractions 22-26 contain 44 g. of C-076 B1a and 0.13 g. of C-076
B1b; fractions 27-30 contain 10.2 g. of C-076 B1a and 0.8 g. of
C-076 B1b.
PREPARATION 8
A mixture of all 8 C-076 components are chromatographed on a high
pressure liquid chromatography column 4 mm..times.30 cm. packed
with 10 micron .mu. Bondapak C.sub.18 silica gel (available from
Waters Associates Inc., Maple Street, Milford, Massachusetts 01757)
eluting with 85:15 (v/v) methanol:water at a constant 40.degree. C.
At a flow rate of 1.2 ml. per minute all eight compounds are
separated and the elution volumes, which under the foregoing
constant conditions are characteristic of the individual compounds
are as follows:
______________________________________ Elution Volume (Ve) Ml
______________________________________ C-076 B.sub.2 b 5.90 C-076
B.sub.2 a 6.52 C-076 A.sub.2 b 7.12 C-076 A.sub.2 a 7.88 C-076
B.sub.1 b 8.36 C-076 B.sub.1 a 9.60 C-076 A.sub.1 b 10.24 C-076
A.sub.1 a 11.88 ______________________________________
The separation of C-076 "b" components from the respective "a"
components is accomplished using techniques such as high pressure
liquid chromatography. An absolute methanol solution of 30
microliters of a mixture of C-076 A1a and A1b, estimated to contain
30 micrograms of C-076 A1b is placed on a 3.times.250 mm. high
pressure liquid chromatography column containing Spherisorb 5
micron ODS (available from Spectra Physics) as packing. The column
is eluted with 85:15 methanol-water at a rate of 0.15 ml./min. The
elution of the products are followed by observing the ultraviolet
absorption of the eluent and collecting the individual components
at the outlet of the UV monitor. 30 Micrograms of C-076 A1b is
recovered in this manner.
* * * * *